Vaccines

ABSTRACT

The present invention provides vaccine compositions comprising an oil-in-water emulsion optionally with 3 De-O-acylated monophosphoryl lipid A and QS21. The vaccines compositions are potent inducers of a range of immune responses.

[0001] The present invention relates to novel vaccine formulations, tomethods of their production and to their use in medicine. In particular,the present invention relates to an oil in water emulsion. Suchemulsions comprise tocopherol, squalene, Tween 80, Span 85 and Lecithinand have useful adjuvant properties. Vaccines containing QS21, an Hplcpurified non-toxic fraction derived from the bark of Quillaja SaponariaMolina, and/or 3 De-O-acylated monophosphoryl lipid A (3 D-MPL),together with such oil in water emulsions also form part of theinvention.

[0002] 3 De-O-acylated monophosphoryl lipid A is known from GB2220 211(Ribi). Chemically it is a mixture of 3 De-O-acylated monophosphoryllipid A with 4, 5 or 6 acylated chains and is manufactured by RibiImmunochem Montana. A preferred form of 3 De-O-acylated monophosphoryllipid A is disclosed in International Patent Application No. 92/116556.

[0003] QS21 is a Hplc purified non toxic fraction of a saponin from thebark of the South American tree Quillaja Saponaria Molina and its methodof its production is disclosed (as QA21) in U.S. Pat. No. 5,057,540.

[0004] Oil in water emulsions per se are known in the art, and have beensuggested to be useful as adjuvant compositions (EPO 399843).

[0005] The present invention is based on the surprising discovery thatan oil in water emulsion of the present invention, which unlikeemulsions of the prior art contain tocopherol, as such or in combinationwith QS21 and/or 3 D-MPL enhance immune responses to a given antigen.Such enhancement available affords better immunological responses thanhitherto before.

[0006] Additionally the oil in water emulsions of the present inventionwhen formulated with 3 D-MPL and QS21 are preferential stimulators ofIgG2a production and TH1 cell response. This is advantageous, because ofthe known implication of TH₁ response in cell mediated response. Indeedin mice induction of IgG2a is correlated with such an immune response.

[0007] For example a vaccine formulation of the HIV antigen gp120 insuch a combination results in a powerful synergistic induction of gp120protein specific immune responses.

[0008] The observation that it is possible to induce strong cytolytic Tlymphocyte responses is significant as these responses, in certainanimal models have been shown to induce protection against disease.

[0009] The present inventors have shown that the combination of theadjuvants QS21 and 3D-MPL together with an oil in water emulsion with anantigen results in a powerful induction of CS protein specific CTL inthe spleen. QS21 also enhances induction of CTL on its own, while 3D-MPLdoes not.

[0010] Induction of CTL is easily seen when the target antigen issynthesised intracellularly (e.g. in infections by viruses,intracellular bacteria, or in tumours), because peptides generated byproteolytic breakdown of the antigen can enter the appropriateprocessing pathway, leading to presentation in association with class Imolecules on the cell membrane. However, in general, pre-formed solubleantigen does not reach this processing and presentation pathway, anddoes not elicit class I restricted CTL. Therefore conventionalnon-living vaccines, while eliciting antibody and T helper responses, donot generally induce CTL mediated Immunity. The combination of the twoadjuvants QS21 and 3D-MPL together with an oil in water emulsion canovercome this serious limitation of vaccines based or recombinantproteins, and induce a wider spectrum of immune responses.

[0011] CTL specific for CS protein have been shown to protect frommalaria in mouse model systems (Romero et al. Nature 341:323 (1989)). Inhuman trials where volunteers were immunised using irradiatedsporozoites of P. falciparum, and shown to be protected againstsubsequent malaria challenge, induction of CTL specific for CS epitopeswas demonstrated (Malik et al. Proc. Natl. Acad. Sci. USA 88:3300(1991)).

[0012] The ability to induce CTL specific for an antigen administered asa recombinant molecules is relevant to malaria vaccine development,since the use of irradiated sporozoites would be impractical, on thegrounds of production and the nature of the immune response.

[0013] RTS is a hybrid protein comprising substantially all theC-terminal portion of the circumsporozoite (CS) protein of P.falciparumlinked via four amino acids of the preS₂ portion of Hepatitis B surfaceantigen to the surface (S) antigen of hepatitis B virus. It's fillstructure is disclosed in co-pending International Patent ApplicationNo. PCT/EP92/02591, published under Number WO 93/10152 claiming priorityfrom UK patent application No.9124390.7. When expressed in yeast RTS isproduced as a lipoprotein particle, and when it is co-expressed with theS antigen from HBV it produces a mixed particle known as RTS,S.

[0014] In addition to human immunodeficiency virus and malaria vaccines,the ability to induce CTL responses would benefit vaccines againstherpes simplex virus, cytomegalovirus, and generally all cases where thepathogen has an intracellular life stage.

[0015] Likewise, CTL specific for known tumour antigens could be inducedby a combination of a recombinant tumour antigen and the two adjuvants.This would allow the development of anti cancer vaccines.

[0016] In certain systems, the combination of 3D-MPL and QS21 togetherwith an oil in water emulsion have been able to synergistically enhanceinterferon γ production. The present inventors have demonstrated thepotential of 3D-MPL and QS21 together with an oil in water emulsion byutilising a herpes simplex antigen known as gD₂t. gD₂t is a solubletruncated glycoprotein D from HSV-2 and is produced in CHO cellsaccording to the methodology Berman et al. Science 222 524-527.

[0017] IFN-γ secretion is associated with protective responses againstintracellular pathogens, including parasites, bacteria and viruses.Activation of macrophages by IFN-γ enhances intracellular killing ofmicrobes and increases expression of Fc receptors. Direct cytotoxicitymay also occur, especially in synergism with lymphotoxin (anotherproduct of TH1 cells). IFN-γ is also both an inducer and a product of NKcells, which are major innate effectors of protection. TH1 typeresponses, either through IFN-γ or other mechanisms, providepreferential help for IgG2a immunoglobulin isotypes.

[0018] Glycoprotein D is located on the viral envelope, and is alsofound in the cytoplasm of infected cells (Eisenberg R. J. et al J. ofVirol. 1980 35 428-435). It comprises 393 amino acids including a signalpeptide and has a molecular weight of approximately 60 kD. Of all theHSV envelope glycoproteins this is probably the best characterized(Cohen et al. J. Virology 60 157-166). it is known to play a centralrole in viral attachment to cell membranes. Moreover, glycoprotein D hasbeen shown to be able to elict neutralizing antibodies in vivo (Eing etal. J. Med Virology 127: 59-65). However, latent HSV2 virus can still bereactivated and induce recurrence of the disease despite the presence ofhigh neutralizing antibodies titre in the patients sera It is thereforeapparent that the ability to induce neutralizing antibody alone isinsufficient to adequately control the disease.

[0019] In order to prevent recurrence of the disease, any vaccine willneed to stimulate not only neutralizing antibody, but also cellularimmunity mediated through T-cells, particularly cytotoxic T-cells.

[0020] In this instance the gD₂t is HSV2 glycoprotein D of 308 aminoacids which comprises amino acids 1 though 306 of the naturallyoccurring glycoprotein with the addition of Asparagine and Glutamine atthe C terminal end of the truncated protein. This form of the proteinincludes the signal peptide which is cleaved to yield a mature 283 aminoacid protein. The production of such a protein in Chinese Hamster ovarycells has been described in Genentech's European patent EP-B-139 417.

[0021] The mature truncated glycoprotein D (rgD2t) or equivalentproteins secreted from mammalian cells, is preferably used in thevaccine formulations of the present invention.

[0022] The formulations of the present invention are very effective ininducing protective immunity in a genital herpes model in guinea pigs.Even with very low doses of antigen (e.g. as low as 5 μg rgD2t) theformulations protect guinea pigs against primary infection and alsostimulate specific neutralising antibody responses. The inventors,utilising formulation of the present invention, have also demonstratedEffector cell mediated responses of the TH1 type in mice.

[0023] Accordingly, in one preferred embodiment of the present inventionprovides a vaccine or pharmaceutical formulation comprising an antigenin conjunction with 3 De-O-acylated monophosphoryl lipid A, QS21and anoil in water emulsion wherein the oil in water emulsion comprises ametabolisible oil, such as squalene, alpha tocopherol and tween 80. Sucha formulation is suitable for a broad range of monovalent or polyvalentvaccines. Additionally the oil in water emulsion may contain span 85. Apreferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed inInternational patent application published under No. 92116556—SmithKlineBeecham Biologicals s.a.

[0024] The oil in water emulsion may be utilised on its own or withother adjuvants or immuno-stimulants and therefore an importantembodiment of the invention is an oil in water formulation comprisingsqualene or another metabolisable oil, alpha tocopherol, and tween 80.The oil in water emulsion may also contain span 85 and/or Lecithin.

[0025] Preferably the vaccine formulations will contain an antigen orantigenic composition capable of eliciting an immune response against ahuman or animal pathogen, which antigen or antigenic composition isderived from HIV-1, (such as gp120 or gp160), any of FelineImmunodeficiency virus, human or animal herpes viruses, such as gD orderivatives thereof or Immediate Early protein such as ICP27 from HSV1or HSV2, cytomegalovirus ((esp Human)(such as gB or derivativesthereof), Varicella Zoster Virus (such as gpI, II or III), or from ahepatitis virus such as hepatitis B virus for example Hepatitis BSurface antigen or a derivative thereof, hepatitis A virus, hepatitis Cvirus and hepatitis E virus, or from other viral pathogens, such asRespiratory Syncytial virus, human papilloma virus or Influenza virus,or derived from bacterial pathogens such as Salmonella, Neisseria,Borrelia (for example OspA or OspB or derivatives thereof), orChlamydia, or Bordetella for example P.69, PT and FHA, or derived fromparasites such as plasmodium or Toxoplasma.

[0026] The formulations may also contain an anti-tumour antigen and beuseful for immunotherapeutically treating cancers.

[0027] In an immunotherapeutic animal model for B cell lymphoma, whereBCL-1 mouse lymphoma cells are adminstered intaperitonelly to Balb/cmice on day 0, and mice are vaccinated on days 3, 10 and 20 with theBCL-1 ldlotype, formulation SB62/MPL/QS21 stands out as the most potent,both with respect to antibody titers, and with respect to survival (theonly group with 100% survival). Similarly the ability of thisformulation to stimulate cytotoxic T lymphocytes to the antigensincluded make them a good candidate for formulation of cancer antigens(eg melanoma antigens MAGE-1 and MAGE-3 for immunotherapy of tumors byactive vaccination).

[0028] The formulation may also be useful for utilising with herpeticlight particles such as described in International Patent ApplicationNo. PCT/GB92/00824 and, International Patent Application No.PCT/GB92/00179.

[0029] Derivatives of Hepatitis B Surface antigen are well known in theart and include, inter alia, those PreS₁, PreS₂ S antigens set forthdescribed in European Patent applications EP-A-414 374; EP-A-0304 578,and EP 198-474. In one preferred aspect the vaccine formulation of theinvention comprises the HIV-1 antigen, gp120, especially when expressedin CHO cells. In a further embodiment, the vaccine formulation of theinvention comprises gD₂t as hereinabove defined.

[0030] In a further aspect of the present invention there is provided avaccine as herein described for use in medicine.

[0031] The ratio of QS21:3D-MPL will typically be in the order of 1:10to 10:1; preferably 1:5 to 5:1 and often substantially 1:1. Thepreferred range for optimal synergy is 2.5:1 to 1:1 3D MPL:QS21.Typically for human administration QS21 and 3D MPL will be present in avaccine in the range 1 μg-100 μg, preferably 10 μg-50 μg per dose.Typically the oil in water will comprise from 2 to 10% squalene, from 2to 10% alpha tocopherol and from 0.3 to 3% tween 80. Preferably theratio of squalene: alpha tocopherol is equal or less than 1 as thisprovides a more stable emulsion. Span 85 may also be present at a levelof 1%. In some cases it may be advantageous that the vaccines of thepresent invention will further contain a stabiliser.

[0032] Vaccine preparation is generally described in New Trends andDevelopments in Vaccines, edited by Voller et al., University ParkPress, Baltimore, Md., U.S.A. 1978. Encapsulation within liposomes isdescribed, for example, by Fullerton, U.S. Pat. No. 4,235,877.Conjugation of proteins to macromolecules is disclosed, for example, byLikhite, U.S. Pat. No. 4,372,945 and by Armor et al., U.S. Pat. No.4,474,757.

[0033] The amount of protein in each vaccine dose is selected as anamount which induces an immunoprotective response without significant,adverse side effects in typical vaccinees. Such amount will varydepending upon which specific immunogen is employed and how it ispresented. Generally, it is expected that each dose will comprise 1-1000μg of protein, preferably 2-100 μg, most preferably 4-40 μg. An optimalamount for a particular vaccine can be ascertained by standard studiesinvolving observation of appropriate immune responses in subjects.Following an initial vaccination, subjects may receive one or severalbooster immunisation adequately spaced.

[0034] The formulations of the present invention maybe used for bothprophylatic and therapeutic purposes.

[0035] Accordingly in one aspect, the invention provides a method oftreatment comprising administering an effective amount of a vaccine ofthe present invention to a patient.

[0036] The following examples illustrate the invention.

EXAMPLES cl Example 1

[0037] Vaccine Formulation Comprising the gp120 Antigen of HIV-1.

[0038] The two adjuvant formulations were made each comprising thefollowing oil in water emulsion component.

[0039] SB26: 5% squalene 5% tocopherol 0.4% tween 80; the particle sizewas 500 nm size

[0040] SB62: 5% Squalene 5% tocopherol 2.0% tween 80; the particle sizewas 180 nm

[0041] 1(a) Preparation of Emulsion SB62 (2 Fold Concentrate)

[0042] Tween 80 is dissolved in phosphate buffered saline (PBS) to givea 2% solution in the PBS. To provide 100 ml two fold concentrateemulsion 5 g of DL alpha tocopherol and 5 ml of squalene are vortexed tomix thoroughly. 90 ml of PBS/Tween solution is added and mixedthoroughly. The resulting emulsion is then passed through a syringe andfinally microfluidised by using an M110S microfluidics machine. Theresulting oil droplets have a size of approximately 180 nm.

[0043] 1(b) Preparation of Emulsion SB26

[0044] This emulsion was prepared in an analogous manner utilising 0.4%tween 80.

[0045] 1(c) Other Emulsions as Depicted in Table 1 were Made in anAnalogous Manner. These are tested in the experiments as detailed in thefollowing examples.

[0046] 1(d) Preparation of gp 120 QS21/3D MPL Oil in Water Formulation.

[0047] To the emulsion of 1 a) or b) or c) an equal volume of twiceconcentrated rgp120 (either 20 μg or 100 μg) was added and mixed. Thiswas combined with 50 μg/ml of 3D-MPL and 20 μg/ml of QS21 to give thefinal formulation. Buffer was sed according to salt content and pH.

[0048] Table 3 shows the effectiveness of SB26, utilising gp120 from HIVand 50 μg/ml 3D MPL (MPL) and 20 μg/ml of QS21. The results show thegeometric mean titre (GMT) after the second (P11) and third (P111)inoculations as well as cell mediated responses (CMI) to lymphocyteprolipheration and γ interferon production.

Example 2

[0049] Introduction: Evaluation of an HIV gp 120 Emulsion System

[0050] In this experiment, four emulsions are compared [SB26, SB 62,SB40, SB61]. The influence of each formulation's component (antigen,emulsion, 3D- MPL, QS21) is evaluated.

[0051] 2(b) Groups of Animals Utilised

[0052] There are 22 groups of 5 animals each group received a differentvaccine formulation. - gr 1-4: gp 120 (10 μg) / no emuls ± [3D-MPL,QS21] - gr 5-9: gp 120 (10 μg) / SB26 ± [3D-MPL, QS21] - gr 10: noantigen / SB26 ± [3D-MPL, QS21] - gr 11-12: gp 120 (10 μg) / SB62 ±[3D-MPL, QS21] - gr 13-16: gp 120 (10 μg) / SB40 ± [3D-MPL, QS21] - gr17-20: gp 120 (10 μg) / SB61 ± [3D-MPL, QS21] - gr 21-22: gp 120 (5 μg)/ SB26 ± [3D-MPL, QS21]

[0053] Assays:—antibody titers to gp 120W61D and isotype analysis (allgroups)

[0054] 2(c) Immunization and Bleeding Schedule

[0055] animals were immunized with gp 120W61D, formulated in differento/w emulsions in the presence of 5 μg 3D-MPL and 5 μg QS21 per dose.Negative controls received the equivalent formulations without anyantigen.

[0056] animals were immunized subcutaneously at day 0 and 14. Eachinjection dose was administered in a 100 μl volume.

[0057] blood samples were obtained before Immunization (day 0) and afterImmunization on days 14 (post I), 21 and 28 (7 and 14d. post II).

[0058] 2(d) Analysis of the serological response:

[0059] the 14 days post I and post II serological response was evaluatedin a direct ELISA assay to gp 120W61D.

[0060] the 14 days post II response was also characterized regarding theisotypes of gp 120W61D specific antibodies induced in mice afterimmunization.

[0061] 3 Results and Discussion:

[0062] The results are depicted on Table 2

[0063] a) Comparison of Emulsions in the Presence or Absence of3D-MPL/QS21:

[0064] Addition of emulsions SB26, SB40 or SB62 to the antigen induceshigher antibody titers; In the absence of immunostimulants, the gp 120specific antibodies are essentially IgG1.

[0065] Addition of immunostimulants 3D-MPL and QS21 induces a hugeserological response and a shift of antibodies from IgG1 type toIgG2a/IgG2b: This correlated with cell mediated immunity.

[0066] The preferred combination is [SB26+MPL+QS21].

[0067] c) gp120/SB26 Formulation:

[0068] No significant difference in serological response is observedbetween group 8 and group 9: addition of the gp 120 before or after theother components of the formulation.

[0069] d) Antigen Dose:

[0070] Both 5 and 10 μg of gp 120 formulated in SB26 induce highserological response (groups 5-8 and 21-22).

Example 3

[0071] HSV rgD₂t Formulation

[0072] In analogous manner to that set forth in Example 1a) formulationcomprising the herpes simplex antigen rgD₂t was made and used tovaccinate guinea pigs. Such formulation induced protection against bothrecurrent and initial disease in the guinea pig model.

Example 4

[0073] Screening of Adjuvants for Induction of Protective Anti LymphomaResponses Using Idiotype as Immunogen.

[0074] Therapeutic vaccination of Balb/c mice with idiotype from BCL1lymphoma cells.

[0075] A review of the BALB/C B-cell lymphoma model is discussed byYefenoh et al. Current opinions Immunobiology 1993 5:740-744.

[0076] Groups of 10 mice are injected (ip) with 10⁴ tumor cells at day0, and vaccinated with 100 μg of KLH-coupled immunoglobulin directedagainst BCL1 epitoped (ratio of KLH/1 g: 1/1), in different adjuvantformulations at days 3, 10, 20 (sc immunization in the back). Level ofserum antibodies to KLH and to idiotype, as well as mouse death aremonitored.

[0077] Formulations tested: group # adjuvant MPL: 10 μg  1 none (noantigen) QS21: 10 μg  2 none  3 Freund  4 Alum  5 Alum/MPL  6Alum/MPL/QS21  7 QS21  8 MPL/QS21  9 SB62MPL 10 SB62/MPL/QS21

[0078] groups 12-15: different adjuvants without antigen

[0079] Formulations 8, 9, 10, behaved consisently better as compared tothe others. Formulation 10 stands out as the most potent, both withrespect to antibody titers, and with respect to survival (the only groupwith 100% survival).

Example 5

[0080] Various Formulations of RTS,S

[0081] a) Evaluated in Monkeys

[0082] RTS,S is described in International patent application no.WO93/10152 and was formulated for vaccination of Rheusus monkeys. Fiveanimals were in each group: Group I RTS,S, 3D-MPL (50 μ), AL(OH)₃ GroupII RTS,S, QS21 (20 μ), AL(OH)₃ Group III RTS,S, 3D-MPL (50 μ), QS21(20μ) Group IV RTS,S, 3D-MPL (50 μ), QS21 AL(OH)₃ Group V RTS,S, 3D-MPL (10μ), QS21 AL(OH)₃ Group VI RTS,S, 3D-MPL (50 μ), QS21 SB60

[0083] The animals were inoculated and bled at 14 days post firstimmunisation and 12 days post second immunisation and tested for Antihepatitis B surface antigen immunoglobulin. As can be seen from FIG. 1,animals receiving RTS,S, in SB60 had antibody titres almost six foldhigher than any other group.

[0084] b) Various Formulations of RTS,S—Evaluated in Mice

[0085] 7 groups of animals received the following formulations Group 1RTS,S SB62 Group 2 RTS,S QS21 3D-MPL Group 3 RTS,S QS21 3D-MPL SB62Group 4 RTS,S 3D-MPL Al(OH)₃ Group 5 RTS,S Al(OH)₃ Group 6 Plain Group 7Negative control

[0086] (RTS,S—5 μg/dose, 3 D-MPL 5 μg/dose QS21 5 μg/dose)

[0087] The animals were inoculated and bled at 15 days post firstimmunisation and at day 7 and 15 post second immunisation and assayedfor anti HBSAg antibody subtype. As can be seen from FIG. 2, theemulsion SB62 when formulated with QS21 and 3D-MPL enhancespreferentially and in a synergistic fashion the IgG2a antibody responsewhile SB 62 alone or 3 D-MPL/QS21induce a poor I gG2a response.

Example 6

[0088] Evaluation of Different B Burgdorferi OspA Formulations

[0089] 6.1 Evaluation of Different Formulations of B Burgdorferi ZS7 OspA Lipoproteins.

[0090] OspA lipoprotein for B burgdorferi is described in EuropeanPatent Application 0418 827 Max Plank et al.

[0091] The following formulations were tested in balb/c mice

[0092] 1. OspA+Al(OH)₃

[0093] 2. OspA+Al(OH)₃+3D-MPL (10μ)

[0094] 3. OspA+Al(OH)₃+3D-MPL (30μ)

[0095] 4. OspA+Al(OH)₃+3D-MPL (10μ)+QS21 (5μ)

[0096] 5. OspA+Al(OH)₃+3D-MPL (30μ)+QS21 (15μ)

[0097] 6. OspA+SB60+3D-MPL (10μ)+QS21 (5μ)

[0098] 7. OspA+SB60+3D-MPL (30μ)+QS21 (15μ)

[0099] and antibody titres and sub types studied seven days following afirst inoculation and seven days post second inoculation (inoculationswere at day 0, and 14).

[0100] The results depicted graphically in FIGS. 3 and 4 and show thatthe formulations of the present invention induce high levels ofantibodies and these are preferentially of the IgG2a subtype.

Example 7

[0101] a) HSV-2 ICP 27

[0102] Female Balb/c mice were immunized on day 0 and day 14 in the hindfoot-pads with various formulations of NS1-ICP27. Each injectioncontained 5 μg of NS1-ICP27 and combinations of SB26 oil-in-wateremulsion, QS21 (10 μg) and MPL (25 μg).

[0103] Popliteal lymphnode cells were obtained on day 28 and stimulatedin vitro with syngeneic P815 cells transfected with the ICP27 gene. Thecultures were then tested for specific cytolytic activity on P815 targetcells transfected with ICP27 and P815 ICP27 negative controls.

[0104] Specific lysis results at different effector:target (E:T) ratiosfor different immunization groups were as follows:

[0105] ICP 27 (5 μg)

[0106] E:T P815 P815 transfected with ICP 27 clone 121

[0107] 100:1 −1 0

[0108] 30:1 −2 −3

[0109] 10:1 3 0

[0110] 3:1 1 0

[0111] 1:1 2 2

[0112] 0.3:1 2 2

[0113] ICP 27 (5 μg)+MPL (25 μg)

[0114] E:T P815 P815 transfected with ICP 27 clone 121

[0115] 100:1 5 7

[0116] 30:1 2 2

[0117] 10:1 1 2

[0118] 3:1 −1 −1

[0119] 1:1 −2 −2

[0120] 0.3:1 −4 −1

[0121] ICP 27 (5 μg)+QS21 (10 μg)

[0122] E:T P815 P815 transfected with ICP 27 clone 121

[0123] 100:1 4 17

[0124] 30:1 5 10

[0125] 10:1 3 7

[0126] 3:1 4 5

[0127] 1:1 3 5

[0128] 0.3:1 0 1

[0129] ICP 27 (5 μg)+SB26

[0130] E:T P815 P815 transfected with ICP 27 clone 121

[0131] 100:1 5 20

[0132] 30:1 1 19

[0133] 10:1 2 12

[0134] 3:1 −2 7

[0135] 1:1 1 5

[0136] 0.3:1 1 2

[0137] ICP 27 (5 μg)+MPL (25 μg)+QS21 (10 μg)

[0138] E:T P815 P815 transfected with ICP 27 clone 121

[0139] 100:1 4 13

[0140] 30:1 5 12

[0141] 10:1 4 17

[0142] 3:1 1 3

[0143] 1:1 0 3

[0144] 0.3:1 −1 −2

[0145] ICP 27 (5 μg)+MPL (25 μg)+QS21 (10 μg)+SB26

[0146] E:T P815 P815 transfectes avec ICP27 clone 121

[0147] 100:1 2 20

[0148] 30:1 0 17

[0149] 10:1 3 19

[0150] 3:1 3 8

[0151] 1:1 1 6

[0152] 0.3:1 2 3

[0153] Low ICP27 specific % lysis was obtained in immunization groups:

[0154] ICP 27 (5 μg)+QS21 (10 μg)

[0155] ICP 27 (5 μg)+SB26

[0156] ICP 27 (5 μg)+MPL (25 μg)+QS21 (10 μg)

[0157] ICP 27 (5 μg)+MPL (25 μg)+QS21 (10 μg)+SB26

[0158] while

[0159] ICP 27 (5 μg)

[0160] ICP 27 (5 μg)+MPL (25 μg)

[0161] were negative.

[0162] Thus these data show induction of CTL by recombinant NS1-ICP27 inoil-in-water emulsion alone or with QS21 and MPL; or with QS2 1.

[0163] b) Groups of 5 Balb/c mice were vaccinated in the footpad withthe different vaccines (NS1-1CP27/NS1-ICP27 MPL+QS21/NS1-ICP27 SB26=MPLand QS21/adjuvant alone). One dose contained 10 μg NS1-ICP27, 10 μg MPLand 10 kg QS21.

[0164] Two vaccinations were given at days 0 and 7. Mice were challengedat day 14 with 5.2 10³ TCID50 of HSV2 strain MS. The appearance ofzosteriform lesions and deaths were recorded until day 14 postchallenge.

[0165] ICP27 of HSV2 was expressed in E coli as a fusion protein withNS1 fragment of influenza virus. The protective efficacy of the purifiedrecombinant protein was evaluated in the murine zosteriformi model, incombination with MPL QS21 formulations. Balb/c mice given twovaccinations with NS1-ICP27 combined either with MPL+QS21 or with an oilin water emulsion (SB26)+MPL and QS21 were completely protected againstdisease (no zosteriform lesions) and death following HSV2 wild typechallenge. In contrast, protection was not observed in the micevaccinated either with NS1-ICP27 alone or with NS1-CP27 combined withSB26 without MPL and QS21. TABLE 1 Vehicles two fold concentratedEmulsions SB Tocopherol % Squalene % Tween 80% Span 85% Lecithin % Size26 5 5 0.4 0 0 500 nm 90-100%  800 nm 10-0% 26.1 5 5 0.4 0 0.1 500 nm 635 5 0.6 0 0 500 nm 64 5 5 0.8 0 0 500 nm 61 5 5 1 0 0 250-300 nm 62 5 52 0 0 180 nm 40 5 5 0.4 1 0 500 nm 80-100%  800 nm 20-0% 40.1 5 5 0.4 10.1 500 nm 60 5 5 1 1 0 300 nm 65 5 5 0.4 1.5 0 500 nm 66 5 5 0.4 2 0500 nm

[0166] TABLE 2 HIV gp 120W61D/MOUSE IMMUNOGENICITY (94243)/BALB/C (F.P.)GROUPS IMMUNOGEN (dose)/FORMULATION ELISA TITERS (7 days PII) % IgG1 %IgG2a % IgG2b 1 gP120 10 μg 494 100 0 0 2 gP120 10 μg + 3D-MPL 5 μg 416454 15 32 3 gP120 10 μg + QS21 5 μg 21515 89 4 8 4 gP120 10 μg + 3D-MPL +QS21 52749 22 60 18 5 gP120 10 μg/SB26 12205 94 2 4 6 gP120 10 μg/SB26 +3D-MPL 87388 31 42 27 7 gP120 10 μg/SB26 + QS21 51020 73 15 13 8 gP12010 μg/SB26 + 3D-MPL + QS21 178169 23 57 21 9 SB26 + 3D-MPL + QS21/gP12010 μg 185704 22 60 19 11 gP120 10 μg/SB62 10348 92 8 0 12 gP120 10μg/SB62 + 3D-MPL + QS21 21739 54 37 9 13 gP120 10 μg/SB40 36320 90 7 414 gP120 10 μg/SB40 + 3D-MPL 285219 31 44 25 15 gP120 10 μg/SB40 +QS2148953 78 15 7 16 gP120 10 μg/SB40 + 3D-MPL + QS21 209217 14 67 18 17gP120 10 μg/SB61 <50 — — — 18 gP120 10 μg/SB61 + 3D-MPL 77515 31 50 1919 gP120 10 μg/SB61 + QS21 40737 74 13 13 20 gP120 10 μg/SB61 + 3D-MPL +QS21 59673 29 57 14 21 gP120 5 μg/SB26 25089 99 0 1 22 gP120 5 μg/SB26 +3D-MPL + QS21 242736 18 61 21

[0167] TABLE 3 3D-MPL based forulations: HIV project Monkey studies GMTELisa GMT CMI in Read-Out W61 D Neut. MN DTH in vitro Formulation P11P111 P11 P111 vivo LP IL-2 γIFN gp120 (100 μg)/o/w + 60523 934101:500 >1:3200  + ND + MPL + QS21 gp120 (20 μg)/o/w + 52026 50150 1:5001:2400 + ND + MPL + QS21 “Historical” gp120 (100 20064 μg) o/w + MPL inguinea pigs

1. A vaccine composition comprising an antigen and/or antigeniccomposition, QS21, 3 De-O-acylated monophosphoryl lipid A (3D-MPL) andan oil in water emulsion wherein the oil in water emulsion has thefollowing composition: a metabolisible oil, such as squalene, alphatocopherol and tween
 80. 2. A vaccine as claimed in claim 1 wherein theratio of QS21:3D-MPL is from 1:10 to 10:1.
 3. A vaccine composition asclaimed in claim 1 or 2 capable of invoking a cytolytic T cell responsein a mammal to the antigen or antigenic composition.
 4. A vaccinecomposition as claimed in any of claims 1 to 3 capable of stimulatinginterferon γ production.
 5. A vaccine composition as claimed in any ofclaims 1 to 4 wherein the ratio of QS21:3D-MPL is from 1:1 to 1:2.5. 6.A vaccine composition as claimed herein comprising an antigen orantigenic composition derived from any of Human Immunodeficiency Virus,Feline Immunodeficiency Virus, Herpes Simplex Virus type 1, HerpesSimplex virus type 2, Human cytomegalovirus, Hepatitis A,B,C or E,Respiratory Syncytial virus, human papilloma virus, Influenza virus,Salmonella, Neisseria, Borrelia, Chlamydia, Bordetella, Plasmodium orToxoplasma.
 7. A vaccine as claimed in any of claim 1 to 5 wherein theantigen is a tumour antigen.
 8. Use of composition as defined in any ofclaims 1 to 5 for the manufacture of a vaccine for the prophylatictreatment of viral, bacterial, or parasitic infections.
 9. Use ofcomposition as defined in any of claims 1 to 5 for the manufacture of avaccine for the immunotherapeutic treatment of viral, bacterial,parasitic infections or cancer.
 10. A method of treating a mammalsuffering from or susceptible to a pathogenic infection comprising theadministration of a safe and effective amount of a composition accordingto any of claims 1 to
 5. 11. A method of treating a mammal sufferingfrom cancer comprising the administration of a safe and effective amountof a composition according to any of claims 1 to
 5. 12. A process formaking a vaccine composition according to claims 1 to 5 comprisingadmixing QS21, 3D-MPL and the oil in water emulsion as defined in claim1 with an antigen or antigenic composition.
 13. A vaccine compositioncomprising an antigen or antigenic composition in association with anoil in water emulsion which emulsion comprises: a metabolisable oil,alpha tocopherol, and tween 80.